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Thesis (Ph. D.)--Columbia University, 1940. / Vita. Bibliography: p. 44-45.

Clostridium perfringens was found to produce marked changes in its environment during growth. Decrease in electropotential, as well as decrease in pH, correlated with resumption of cellular activity on transfer into a defined medium. Eight amino acids were found to be essential for growth, while the remaining ten amino acids fell into four categories according to the degree of independence the organism had with respect to them. The form of the B-6 vitamin present in the medium had an important influence on the synthesis of several amino acids. The synthesis and degradation of amino acids was investigated further. The enzyme systems involved in these functions could be described as three major groups: those enzymes involved in synthesis of amino acids and amino compounds from glucose, the enzymes responsible for the interconversion of threonine, glycine and serine, and those involved with glutamic acid, proline and arginine metabolism. Uptake of amino acids by both resting and growing cell suspensions was found to be by an efficient, non-energy dependent diffusion mechanism. During growth approximately 5% of the exogenously supplied glucose was assimilated into cell material, primarily as cell wall or nucleic acid components. Molar growth yields from glucose were found to be significantly greater than the theoretical value. The calculated ratio of reduced to oxidized coenzymes indicated a characteristic trend over the logarithmic and stationary growth phases. The relationship between amino acid metabolism, glucose dissimilation and assimilation, and overall metabolite and energy balances has been discussed with a view to understanding the extent of control and influence C. perfringens has on its own metabolic functions and on its environment. / Science, Faculty of / Microbiology and Immunology, Department of / Graduate

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Cobalamin must be metabolized intracellularly in order to bind two enzymes: methionine synthase in cytoplasm and methylmalonyl-CoA mutase in mitochondria. Defects in this process cause different inborn errors of cobalamin metabolism (cblA-cblG and mut). A previous study described a cobalamin-binding protein, in addition to methylmalonyl-CoA mutase, in crude mitochondrial fractions. The amount of [57Co]cobalamin bound to this protein was increased in cblB, mut and cblD variant2 cell lines, compared to control cell lines. In the present study, this protein was identified as transcobalamin (TC). Mitochondrial fractions from a cblB cell line were incubated with anti-TC antibodies, which precipitated the cobalamin-bound protein. Analysis of mitochondrial and cytoplasmic fractions isolated from a chloroquine-incubated cblF cell line showed that isolated mitochondrial fractions contain lysosomal material, suggesting that the identified TC is lysosomal. Quantification of cobalamin-bound TC levels in whole cell extracts showed significant increases in cblB and mut groups compared to control cell lines.

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Utilization of iron by hemoglobin-producing cells is highly efficient. The acquisition of iron from plasma requires the binding of diferric transferrin (Tf) to its cognate receptor (Tf-R) on the erythroid cell membrane, followed by internalization of the Tf - Tf-R complexes via receptor-mediated endocytosis. Through a poorly understood mechanism, iron is targeted to mitochondria, the site of heme biosynthesis. We believe that a direct interaction between iron-containing endosomes and mitochondria is essential for iron transfer to mitochondria and its efficient incorporation into heme. / In order to illustrate the interaction between endosomes and mitochondria, we have employed flow cytometry. Flow cytometry analysis of reticulocytes (erythrocyte precursors which still synthesize hemoglobin) stained with fluorescent dyes specific to mitochondria and endosomes revealed three distinct populations: mitochondria, endosomes and a population labeled with both dyes. This double-labeled population suggests a population composed of endosomes associated with mitochondria. Using non-fluorescent diferric-Tf, we were able to remove the double population, leaving only the endosomal and the mitochondrial population. This finding has confirmed that the double population is the result of the interaction between the two organelles. / Additionally, we established a cell-free assay consisting of fluorescent mitochondria and endosomes isolated from erythroid cells. Using confocal microscopy, we demonstrated a colocalization between the two organelles. We repeated the assay using fluorescent mitochondria and endosomes isolated from HeLa spinner cells. Using the mitochondrial uncoupler CCCP, we were able to significantly reduce the colocalization between the two organelles, indicating that the interaction between the organelles is specific and that the mitochondrial potential is a requirement for organellar interaction. / Based on our results from flow cytometry and confocal microscopy, we conclude that a specific and direct interaction exists between the two organelles.

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No description available.